Integrin α5β1 Activates the NLRP3 Inflammasome by Direct Interaction with a Bacterial Surface Protein  Hye-Kyoung Jun, Sung-Hoon Lee, Hae-Ri Lee, Bong-Kyu Choi  Immunity  Volume 36, Issue 5, Pages 755-768 (May 2012) DOI: 10.1016/j.immuni.2012.05.002 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Td92 and Live T. denticola Induce Caspase-1 Activation and IL-1β Secretion (A–C) THP-1 cells were treated with Td92, live T. denticola, PP4 (an irrelevant recombinant protein), truncated Td92 (Td-G and Td-B domain), or MDP for 1 to 12 hr (6 hr in A and B). MDP-stimulated cells were then pulsed with 2.5 mM ATP for 30 min. Caspase-1 and IL-1β secreted into the culture supernatants (sup) and procaspase-1, proIL-1β, and β-actin in the cell lysates (cell) were detected by immunoblotting (A). The IL-1β concentration in the culture supernatants was analyzed by ELISA (B and C). The data are shown as the means ± SD. ∗p < 0.01 compared to unstimulated cells. (D) THP-1 cells were pretreated with Z-YVAD-fmk or Ac-YVAD-cho for 30 min before stimulation with Td92 for 6 hr. Caspase-1, IL-1β, and β-actin were detected by immunoblotting. See also Figure S1. Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Td92-Induced IL-1β Activation Is Mediated via NLRP3 THP-1 cells were transfected with NLRP3 siRNA (A), ASC siRNA (B), NLRC4 siRNA (C), or control siRNA for 18 to 48 hr. RNA interference was confirmed by real-time qPCR and immunoblotting. siRNA-transfected cells were treated with Td92, MDP, E. coli RNA, or S. typhimurium flagellin for 6 hr (flagellin was delivered into the cytosol using with Profect P1 protein transfection reagent). Caspase-1, IL-1β, and β-actin were detected by immunoblotting. The real-time qPCR data are shown as the means ± SD ∗p < 0.05 and ∗∗p < 0.01 compared to control siRNA-transfected cells. Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 ATP and K+ Efflux Are Involved in Td92-Induced NLRP3 Activation (A) THP-1 cells were stimulated with Td92, live T. denticola, or PP4 for 2 hr. Extracellular ATP concentration was determined using an ATP bioluminescence assay Kit. The data are shown as the means ± SD. ∗p < 0.01 compared to unstimulated cells. (B–E) THP-1 cells were pretreated with oxATP (B), KCl (C), glybenclamide (D), or tolbutamide (E) for 30 min prior to stimulation with Td92 for 6 hr. Caspase-1, IL-1β, procaspase-1, proIL-1β, and β-actin were detected by immunoblotting. Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Td92 Induces Caspase-1-Dependent Cell Death (A and B) THP-1 cells were treated with Td92, live T. denticola, or PP4 for 6 hr. (C) THP-1 cells were treated with Td92 (10 μg/ml) for 1 to 40 hr. (D and E) THP-1 cells were pretreated with KCl, Ac-YVAD-cho, and Z-YVAD-fmk for 30 min prior to stimulation with Td92 for 6 hr. Cell supernatants were evaluated for the release of the cytoplasmic enzyme LDH with the LDH-cytotoxicity assay Kit. The data are shown as the means ± SD. ∗p < 0.01 compared to Td92-treated cells. (F) THP-1 cells were pretreated with oxATP, KCl, Ac-YVAD-cho, and Z-YVAD-fmk for 30 min prior to stimulation with Td92 for 3 hr. The cells were stained with propidium iodide (PI, 20 μM) and Hoechst 33342 dye (10 μM) for 30 min, and images were acquired with a fluorescence microscope. PI-positive cells were counted out of at least 1,000 total cells with the ImageJ software, and the data are presented as the means ± SD of the percentage of PI-positive cells. ∗p < 0.05 compared to unstimulated controls and #p < 0.05 compared to Td92-stimulated cells. Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Td92 Internalization into the Cells Is Not Required for Caspase-1 Activation or IL-1β Induction (A) THP-1 cells were stimulated with Td92 or S. typhimurium flagellin, which was premixed with or without Profect P1 for 3 and 6 hr. Caspase-1, IL-1β, procaspase-1, proIL-1β, and β-actin were detected by immunoblotting. (B) THP-1 cells were incubated with FITC-labeled Td92, which was premixed with or without Profect P1 (top two panels) or THP-1 cells were pretreated with cytochalasin D for 30 min (bottom panel) before FITC-labeled Td92 treatment for 1 or 4 hr. Internalization of Td92 was analyzed by flow cytometry. Paraformaldehyde-fixed THP-1 cells treated with FITC-Td92 were used as a negative control for internalization. The data are presented as the mean fluorescence intensities (MFI). (C and D) THP-1 cells were pretreated with cytochalasin D for 30 min prior to Td92 treatment for 6 hr. Caspase-1, IL-1β, procaspase-1, proIL-1β, and β-actin were detected by immunoblotting (C) and LDH released into the culture supernatants was measured (D). (E) LDH release was measured in the culture supernatants of Td92- or S. typhimurium flagellin-stimulated cells with or without the addition of Profect P1 for 6 hr. The data are presented as the means ± SD ∗p < 0.05 compared to without Profect P1 treatment. Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Integrin α5β1 Directly Interacts with Td92 to Activate Caspase-1 (A) THP-1 cell lysates were reacted with Td92, Td92 preincubated with fibronectin (Fn), or PP4. The reaction mixtures were incubated with Ni-NTA agarose. Agarose-bound proteins were subjected to immunoblotting for the detection of integrin subunits (α5, αν, β1, and β3), fibronectin (Fn), Td92, and PP4. (B) Protein G-agarose was incubated with integrin α5β1 Ab, subsequently, with recombinant human integrin α5β1. After incubating the agarose with Td92, fibronectin (Fn), or PP4, the proteins bound to the integrin α5β1-coated agarose were analyzed by immunoblotting. (C) THP-1 cells were pretreated with cytochalasin D for 30 min and then incubated with FITC-labeled Td92 (10 μg/ml) for 2 hr. After fixation with 3.8% paraformaldehyde, the cells were incubated with an integrin α5β1 Ab and then a Cy3-conjugated secondary Ab with the nuclear staining with Hoechst 33342 and were observed by confocal microscopy (×2000). Scale bars represent 2.5 μm. (D) The THP-1 cells transfected with integrin α5 siRNA were treated with Td92 for 6 hr. Caspase-1, IL-1β, proIL-1β, β-actin, and integrin α5 were detected by immunoblotting. (E) THP-1 cells were incubated with an integrin α5β1 Ab or IgG isotype for 1 hr prior to incubation with FITC-labeled Td92 (10 μg/ml) for 30 min, and Td92 binding was analyzed by flow cytometry. The data are presented as the mean fluorescence intensities (MFI). (F–H) THP-1 cells (F and G) or human PBMC-derived macrophages (H) were pretreated with an integrin α5β1 Ab or IgG isotype for 1 hr prior to stimulation with Td92 for 6 hr. Caspase-1, IL-1β, procaspase-1, proIL-1β, and β-actin were detected by immunoblotting (F and the top panel of H), and the IL-1β concentration in the culture supernatants was measured by ELISA (G and the bottom panel of H). (I) THP-1 cells were pretreated with an integrin α5β1 Ab or IgG isotype for 1 hr before stimulation with Td92 for 2 hr. Extracellular ATP concentrations were determined with an ATP bioluminescence assay Kit. The data are shown as the means ± SD. ∗p < 0.01 compared to unstimulated cells and #p < 0.01 compared to Td92-treated cells (G, H, and I). See also Figure S2. Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 NF-κB Activation Has an Important Role in Td92-Induced proIL-1β Expression and NLRP3 Activation (A) THP-1 cells were pretreated with various inhibitors for 30 min prior to stimulation with Td92 for 6 hr. IL-1β, proIL-1β, and β-actin were detected by immunoblotting. (B) THP-1 cells were pretreated with an integrin α5β1 Ab or IgG isotype for 1 hr prior to stimulation with Td92 for 30 min. The cell lysates were subjected to immunoblotting for IκBα degradation and MAPK phosphorylation. (C–E) THP-1 cells were stimulated with Td92, T. denticola, LPS, TNF-α, or PP4 for 1 hr with or without pretreatment with the integrin α5β1 Ab or oxATP, and NF-κB localization was detected with an anti-human NF-κB p65 Ab and a Cy3-conjugated secondary Ab with nuclear staining with Hoechst 33342. NF-κB translocation into the nucleus of the cells was observed with a confocal laser scanning microscope (C). The number of cells positive for NF-κB activation after pretreatment with the integrin α5β1 Ab (D) or oxATP (E) was counted out of a total of at least 500 cells with the NIH ImageJ software, and the data are presented as the means ± SD. (F) THP-1 cells were pretreated with Bay-117082 for 30 min before stimulation with Td92 or LPS for 6 hr. LPS-stimulated cells were pulsed with 2.5 mM ATP for 30 min. Caspase-1, IL-1β, procaspase-1, proIL-1β, and β-actin were detected by immunoblotting. (G) THP-1 cells were stimulated with Td92 or LPS for 4 hr and NLRP3 mRNA expression was analyzed by real-time qPCR. The data are shown as the means ± SD. (H) THP-1 cells were pretreated with an integrin α5β1 Ab, IgG isotype, Bay-117082 (Bay, 10 μM), or oxATP (Ox, 300 μM) for 30 min or 1 hr prior to stimulation with Td92 for 6 hr. The cells were subjected to immunoblotting for the detection of NLRP3 expression. Fibronectin (Fn)-stimulated cells were also included. (I) The THP-1 cells transfected with MyD88 or TRIF siRNA were treated with Td92 or LPS for 1 hr, and NF-κB localization was detected with an anti-human NF-κB p65 Ab and a Cy3-conjugated secondary Ab with nuclear staining with Hoechst 33342. The data are shown as the means ± SD. ∗∗p < 0.01 and ∗p < 0.05 compared to unstimulated cells (D, E, G, and I); #p < 0.01 compared to Td92-treated cells (D and E) or to control siRNA-transfected cells (I). Scale bars represent 10 μm in (C) and 50 μm in (D), (E), and (I). Immunity 2012 36, 755-768DOI: (10.1016/j.immuni.2012.05.002) Copyright © 2012 Elsevier Inc. Terms and Conditions